Structure-Activity Relationships toward the Identification of a High-Potency Selective Human Toll-like Receptor-7 Agonist.

Toll-like receptor (TLR)-7 agonists are immunostimulatory vaccine adjuvants. A systematic structure-activity relationship (SAR) study of TLR7-active 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine led to the identification of a potent hTLR7-specific p-hydroxymethyl IMDQ 23 with an EC50 value of 0.22 µM. The SAR investigation also resulted in the identification of TLR7 selective carboxamide 12 with EC50 values of 0.32 µM for hTLR7 and 18.25 µM for hTLR8. In the vaccination study, TLR7-specific compound 23 alone or combined with alum (aluminum hydroxide wet gel) showed adjuvant activity for a spike protein immunogen in mice, with enhanced anti-spike antibody production. Interestingly, the adjuvant system comprising carboxamide 12 and alum showed prominent adjuvant activity with high levels of IgG1, IgG2b, and IgG2c in immunized mice, confirming a balanced Th1/Th2 response. In the absence of any apparent toxicity, the TLR7 selective agonists in combination with alum may make a suitable vaccine adjuvant.

Phosphate adsorption on dried alum sludge: Modeling and application to treatment of dairy effluents.

This study evaluates Alum sludge from drinking water treatment plants for the efficient and cost-effective removal of phosphates from aqueous solutions. Extensive characterization and batch experiments have established that optimal phosphate removal was achieved with a sludge dosage of 20 g L-1 (at an initial phosphate concentration of 100 mg L-1), a pH of 5, a temperature of 23 °C, and a stirring speed of 200 rpm. These conditions significantly reduced phosphate levels, ensuring compliance with legal discharge limits. The Langmuir isotherm, pseudo-second-order kinetic and intraparticle diffusion models best described the adsorption process, highlighting the spontaneous and endothermic nature of the phenomenon. The sludge effectively reduced phosphate concentrations to acceptable levels when applied to dairy effluents. This study underscores the potential of Alum sludge as a viable solution for phosphate management in environmental cleanup efforts.

The orientation of CpG conjugation on aluminum oxyhydroxide nanoparticles determines the immunostimulatory effects of combination adjuvants.

In subunit vaccines, aluminum salts (Alum) are commonly used as adjuvants, but with limited cellular immune responses. To overcome this limitation, CpG oligodeoxynucleotides (ODNs) have been used in combination with Alum. However, current combined usage of Alum and CpG is limited to linear mixtures, and the underlying interaction mechanism between CpG and Alum is not well understood. Thus, we propose to chemically conjugate Alum nanoparticles and CpG (with 5' or 3' end exposed) to design combination adjuvants. Our study demonstrates that compared to the 3'-end exposure, the 5'-end exposure of CpG in combination adjuvants (Al-CpG-5') enhances the activation of bone-marrow derived dendritic cells (BMDCs) and promotes Th1 and Th2 cytokine secretion. We used the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen (HBsAg) as model antigens to demonstrate that Al-CpG-5' enhanced antigen-specific antibody production and upregulated cytotoxic T lymphocyte markers. Additionally, Al-CpG-5' allows for coordinated adaptive immune responses even at lower doses of both CpG ODNs and HBsAg antigens, and enhances lymph node transport of antigens and activation of dendritic cells, promoting Tfh cell differentiation and B cell activation. Our novel Alum-CPG strategy points the way towards broadening the use of nanoadjuvants for both prophylactic and therapeutic vaccines.

Remediation of sediments contaminated by harmful heavy metals using aluminum sulfate: Sustainable approach for practical applications.

Chemical washing could be suitable for the remediation of marine sediments contaminated with harmful heavy metals. Considering green and sustainable remediation (GSR), the application of aluminum sulfate (AS) is intended to reduce the costs and environmental impacts. We extracted harmful heavy metals from manganese nodules using an ion exchange mechanism that occurs when AS dissociates in water. AS in the range from 2 % to 5 % was used. The remediation efficiencies using 5 % AS were found to be the highest, at 91.8 % for Ni and ≥ 100 % for other harmful heavy metals. The Pearson's coefficient evaluation showed that increasing elapsed time did not significantly affect the extraction of harmful heavy metals. Pollutants in post-processing products may not cause secondary pollutions if solidification/stabilization and additional treatments are used. Our results can serve as fundamental data for the actual remediation processes using AS not only for deep-sea mining tailings but also contaminated marine sediments.

Phosphorus fractions and speciation in an alkaline, manured soil amended with alum, gypsum, and Epsom salt.

Snowmelt runoff is a dominant pathway of phosphorus (P) losses from agricultural lands in cold climatic regions. Soil amendments effectively reduce P losses from soils by converting P to less soluble forms; however, changes in P speciation in cold climatic regions with fall-applied amendments have not been investigated. This study evaluated P composition in soils from a manured field with fall-amended alum (Al2(SO4)3·18H2O), gypsum (CaSO4·2H2O), or Epsom salt (MgSO4·7H2O) using three complementary methods: sequential P fractionation, scanning electron microscopy with energy-dispersive X-rays (SEM-EDX) spectroscopy, and P K-edge X-ray absorption near-edge structure spectroscopy (XANES). Plots were established in an annual crop field in southern Manitoba, Canada, with unamended and amended (2.5 Mg ha-1) treatments having four replicates in 2020 fall. Soil samples (0-10 cm) taken from each plot soon after spring snowmelt in 2021 were subjected to P fractionation. A composite soil sample for each treatment was analyzed using SEM-EDX and XANES. Alum- and Epsom salt-treated soils had significantly greater residual P fraction with a higher proportion of apatite-like P and a correspondingly lower proportion of P sorbed to calcite (CaCO3) than unamended and gypsum-amended soils. Backscattered electron imaging of SEM-EDX revealed that alum- and Epsom salt-amended treatments had P-enriched microsites frequently associated with aluminum (Al), iron (Fe), magnesium (Mg), and calcium (Ca), which was not observed in other treatments. Induced precipitation of apatite-like species may have been responsible for reduced P loss to snowmelt previously reported with fall application of amendments.

Parametric study of coagulant recovery from water treatment sludge towards water circular economy.

This study aims to recover the used coagulants from two water treatment plants via acidification technique. The water treatment sludge (WTS) was acidified with sulfuric acid (H2SO-4) at variable normalities (0.5, 1, 1.5, 2.0 and 2.5 N). The surface morphology and functionalities of both recovered coagulants were analysed using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR). The performance of recovered coagulants was tested for turbidity removal in surface water treatment at different coagulant dosages and pH. It was found that the optimum normality of H2SO4 for recovered alum was 1.5 N, where 66% turbidity removal was recorded. The recovered PAC treated with 1.0 N H2SO4 indicated high turbidity removal percentage, which was 50.5%. The turbidity removal increased with increasing coagulant dosage. More than 80% turbidity removal was achieved with 40 mg/L dosage of recovered alum and recovered PAC. Maximum removal (85%) was observed with 50 mg/L dosage of recovered alum. For commercial coagulant, the turbidity removal was higher, with a difference of up to 6% in favor of recovered alum. The potential reuse of coagulants can be explored in order to reduce the operating costs and promotes the reduction of WTS disposal.

Alum-anchored intratumoral retention improves the tolerability and antitumor efficacy of type I interferon therapies.

Effective antitumor immunity in mice requires activation of the type I interferon (IFN) response pathway. IFNα and IFNß therapies have proven promising in humans, but suffer from limited efficacy and high toxicity. Intratumoral IFN retention ameliorates systemic toxicity, but given the complexity of IFN signaling, it was unclear whether long-term intratumoral retention of type I IFNs would promote or inhibit antitumor responses. To this end, we compared the efficacy of IFNα and IFNß that exhibit either brief or sustained retention after intratumoral injection in syngeneic mouse tumor models. Significant enhancement in tumor retention, mediated by anchoring these IFNs to coinjected aluminum-hydroxide (alum) particles, greatly improved both their tolerability and efficacy. The improved efficacy of alum-anchored IFNs could be attributed to sustained pleiotropic effects on tumor cells, immune cells, and nonhematopoietic cells. Alum-anchored IFNs achieved high cure rates of B16F10 tumors upon combination with either anti-PD-1 antibody or interleukin-2. Interestingly however, these alternative combination immunotherapies yielded disparate T cell phenotypes and differential resistance to tumor rechallenge, highlighting important distinctions in adaptive memory formation for combinations of type I IFNs with other immunotherapies.

Combination of conserved recombinant proteins (NP & 3M2e) formulated with Alum protected BALB/c mice against influenza A/PR8/H1N1 virus challenge.

PURPOSE: Influenza is one of the most important agents of pandemic outbreak causing substantial morbidity and mortality. Vaccination strategies of influenza must be adapted annually due to constant antigenic changes in various strains. Therefore, the present study was conducted to evaluate protective immunity of the conserved influenza proteins. METHODS: For this purpose, three tandem repeats of M2e (3M2e) and NP were separately expressed in E. coli and were purified using column chromatography. Female Balb/c mice were injected intradermally with a combination of the purified 3M2e and NP alone or formulated with Alum (AlOH3) adjuvant in three doses. The mice were challenged by intranasal administration of H1N1 (A/PR/8/34) 2 weeks after the last vaccination. RESULTS: The results demonstrated that recombinant NP and M2e proteins are immunogenic and could efficiently elicit immune responses in mice compared to non-immunized mice. The combination of 3M2e and NP supplemented with Alum stimulated both NP and M2e-specific antibodies, which were higher than those stimulated by each single antigen plus Alum. In addition, the secretion of IFN-γ and IL-4 as well as the induction of lymphocyte proliferation in mice received the mixture of these proteins with Alum was considerably higher than other groups. Moreover, the highest survival rate (86%) with the least body weight change was observed in the mice immunized with 3M2e and NP supplemented with Alum followed by the mice received NP supplemented with Alum (71%). CONCLUSION: Accordingly, this regimen can be considered as an attractive candidate for global vaccination against influenza.

Formulation of a recombinant HIV-1 polytope candidate vaccine with naloxone/alum mixture: induction of multi-cytokine responses with a higher regulatory mechanism.

The potency of a vaccine highly depends upon the nature of the adjuvant used. There are a variety of ineffective vaccines, such as HIV-1 vaccine candidates, that need to be optimized with new adjuvant formulations to improve vaccine potency and efficacy. Studies show the potency of naloxone (NLX)/alum mixture in the induction of Th1/Th2 response for vaccine. However, other immunologic patterns inducing by this adjuvant and its immunoregulatory effect is unclear. In this regard, the aim of the present study was to investigate the effect of the NLX/alum mixture, as an adjuvant, on cytokine networks and immunoregulatory activity for an HIV-1 polytope vaccine. BALB/c mice were divided into six groups (n = 6) and immunized subcutaneously with 10 µg of the vaccine formulated with NLX/alum, NLX, alum, and Freund's adjuvants. At the same time, the mice in the control groups received an equal volume of PBS or NLX. The lymphocyte proliferation assay was carried out using the BrdU method. ELISA was used to measure the levels of IFN-γ, IL-2, IL-4, IL-10, IL-12, and IL-17 cytokines, total IgG, as well as IgG1 and IgG2a subtypes in serum samples. Our findings showed that mice receiving the NLX/alum-adjuvanted vaccine exhibited increased antibody levels compared with other groups. In addition, there was a considerable difference in the levels of IgG1, IgG2a, IFN-γ, IL-2, IL-10, IL-12, and IL-17 in mice receiving the NLX/alum-adjuvanted vaccine as compared with other groups. The NLX/alum mixture, as an adjuvant, may have a positive effect on the induction of multi-cytokine responses, as well as the increased level of IL-10, showing its higher immunogenicity with a higher immunoregulatory mechanism.

Coagulation Treatment of Wastewater: Kinetics and Natural Coagulant Evaluation.

In this study, three coagulants (ferromagnetite (F), alum (A), and eggshells (E)) and their hybrids (FA, FE, and FEA) were investigated as possible cost-effective coagulants for the treatment of industrial wastewater. Scanning electron microscopy (SEM) coupled with energy-dispersive X-ray (EDX) was used to characterize the morphological and elemental compositions of the coagulants. The effects of coagulant dosage (10-60 mg/L) and settling time were investigated for the removal of turbidity, color, and total suspended solids. A jar tester (JTL6) operating at conditions of 150 rpm for 2 min (rapid mixing) and 30 rpm for 15 min (slow mixing) was employed. Results from the characterized supernatant showed about 80% removal of the contaminants. The prospects of F were proven to be the most effective as compared to the binary (FA > FE) and the ternary hybridized (FEA) coagulants. At an optimum dosage and settling time of 20 mg/L and 30 min, respectively, the treatability performance of F was clearly proven to be viable for wastewater treatment.

Protective effect of a DNA vaccine cocktail encoding ROP13 and GRA14 with Alum nano-adjuvant against Toxoplasma gondii infection in mice.

Toxoplasma gondii is an obligate intracellular protozoan parasite that can cause serious public health problems. The development of a safe and effective vaccine against T. gondii is urgently needed to prevent and control the spread of toxoplasmosis. The aim of this study was to evaluate the immune responses induced by a pcGRA14 + pcROP13 vaccine cocktail in BALB/c mice. All groups were immunized intramuscularly three times at two-week intervals. The production of anti-Toxoplasma gondii lysate antigen (TLA) antibodies, lymphocyte proliferation, serum levels of IFN-γ and IL-4 cytokines and the survival time were monitored after vaccination and challenged with the virulent RH strain of T. gondii. The results showed that immunization with the pcGRA14 + pcROP13 DNA vaccine significantly increased the production of specific IgG antibodies and cytokines against toxoplasmosis. Interestingly, high levels of IgG2a and IFN-γ were found in animals vaccinated with DNA vaccine cocktail. Furthermore, immunized mice challenged with the RH strain of T. gondii showed prolonged survival time when compared to control groups (P <0.05). The present study demonstrates the potential of a DNA cocktail vaccine expressing pcGRA14 and pcROP13 in developing specific immune responses and providing effective protection against T. gondii infection.

Removal efficiency of dissolved organic matter from secondary effluent by coagulation-flocculation processes.

Wastewater reuse has been widely discussed as an essential strategy to minimize the consumption of drinking water for less noble purposes. During biological wastewater treatment, organic matter is converted into a complex matrix containing a variety of soluble organic compounds. The objective of the present study was to evaluate the removal efficiency of the residual organic load in the final effluent from wastewater treatment plant with a conventional activated sludge process by different coagulants and parameters of coagulation-flocculation process, using dissolved organic carbon (DOC) concentration, molecular weight (MW) size distribution by size exclusion chromatography (SEC) coupled to mass spectrometry (MS), and zeta potential (ZP) analyses. The results showed a DOC removal efficiency up to 45% with iron chloride, and of 38% for aluminum sulfate and 31% for PAC coagulants. ZP was also measured during the procedures and authors conclude that the ZP also does not have a determining role in these removals. SEC and MS assessment was able to detect changes on secondary effluent molecular weight distribution profile after effluent coagulation-flocculation, this technique might be a promising tool to understand the composition of effluent organic matter and be helpful to estimate and optimize the performance of wastewater effluents treatment processes.

Robust Immune Responses Elicited by a Hybrid Adjuvant Based on ß-Glucan Particles from Yeast for the Hepatitis B Vaccine.

The use of particulate adjuvants offers an interesting method for enhancing and modulating the immune responses elicited by vaccines. Aluminum salt (Alum) is one of the most important immune adjuvants approved by the Food and Drug Administration for use in humans because of its safety and efficacy, but it lacks the capacity to induce strong cellular and mucosal immune responses. In this study, we designed an antigen delivery system that combines aluminum salts with ß-glucan particles. The ß-glucan-aluminum particles (GP-Al) exhibited a highly uniform size of 2-4 µm and could highly specifically target antigen-presenting cells (APCs) and strongly induce dendritic cell (DC) maturation and cytokine secretion. In vivo studies showed that both WT mice and HBV-Tg mice immunized with hepatitis B surface antigen (HBsAg)-containing GP-Al displayed high anti-HBsAg IgG titers in the serum. Furthermore, in contrast to mice receiving the antigen alone, mice immunized with the particulate adjuvant exhibited IgG2a antibody titers and higher antigen-specific IFN-γ levels in splenocytes. In conclusion, we developed GP-Al microspheres to serve as a hepatitis B vaccine to enhance both humoral and cellular immune responses, representing a safe and promising system for antigen delivery.

Stability of Alum-Containing Paper under Alkaline Conditions.

The present contribution evaluates the methods of degradation and stabilization of alum-containing paper with a focus on the alkaline environment achieved by deacidification procedures. In terms of reviewed subjects, the contribution focuses on alum-rosin sized paper, which is still used as a carrier of knowledge and information; however, it also mentions cellulose itself and other brands of paper. The contribution summarizes the results on the homogeneity of the distribution of alum and rosin in the paper mass and on the paper surface. It provides the knowledge gained in the field of alkaline hydrolysis and oxidation with special regard to transition metal species. It shows the values of alkaline reserves achieved in the main mass-deacidification processes. On the basis of the acquired knowledge, the contribution emphasizes the procedures of paper stabilization. Criteria of "increased mechanical permanence and lifetime prolongation" adopted to evaluate and compare the efficacy of individual mass-deacidification processes were applied and corresponding data are introduced. The contribution also draws attention to the existence of open issues in the area of paper degradation and stabilization.

Particulate Alum via Pickering Emulsion for an Enhanced COVID-19 Vaccine Adjuvant.

For rapid response against the prevailing COVID-19 (coronavirus disease 19), it is a global imperative to exploit the immunogenicity of existing formulations for safe and efficient vaccines. As the most accessible adjuvant, aluminum hydroxide (alum) is still the sole employed adjuvant in most countries. However, alum tends to attach on the membrane rather than entering the dendritic cells (DCs), leading to the absence of intracellular transfer and process of the antigens, and thus limits T-cell-mediated immunity. To address this, alum is packed on the squalene/water interphase is packed, forming an alum-stabilized Pickering emulsion (PAPE). "Inheriting" from alum and squalene, PAPE demonstrates a good biosafety profile. Intriguingly, with the dense array of alum on the oil/water interphase, PAPE not only adsorbs large quantities of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) antigens, but also harbors a higher affinity for DC uptake, which provokes the uptake and cross-presentation of the delivered antigens. Compared with alum-treated groups, more than six times higher antigen-specific antibody titer and three-fold more IFN-γ-secreting T cells are induced, indicating the potent humoral and cellular immune activations. Collectively, the data suggest that PAPE may provide potential insights toward a safe and efficient adjuvant platform for the enhanced COVID-19 vaccinations.

Corneal Thinning Induced by Self-administered Alum Substance: A Case Report and Analysis of the Active Components.

We report a case of severe ocular injury and impaired vision after self-administration of alum. A 56-year-old female administered an alum substance in the left eye and experienced severe corneal thinning, a scar, and decreased vision. The active compounds in the alum substance were analyzed using scanning electron microscopy. When topically administered, alum may cause severe ocular injury. Public awareness, early recognition of the injuries, and timely intervention may prevent permanent ocular damage.

Alum sludge as an efficient sorbent for hydrogen sulfide removal: Experimental, mechanisms and modeling studies.

This paper firstly reported a systematic study of using alum sludge (waterworks residue) for H2S adsorption. Various trials were performed at ambient temperature in a fixed bed column to study the effects of H2S flow rate, sorbent bed depth on the alum sludge adsorption efficiency of H2S. The Breakthrough Curves were simulated by the Thomas model, Bed Depth Service Time model and Yoon-Nelson models. The mechanisms of H2S adsorption onto alum sludge was examined by different physiochemical characterizations of exhausted and raw alum sludge. Moreover, the mass transfer coefficients were determined from mathematical descriptions of breakthrough curves. The alum sludge adsorption capacity was determined to be 374.2 mg of H2S/g, slightly decreasing with the increasing flow rate and increasing with the increasing bed depth. All the three models successfully predict breakthrough curves which could be used for scaling-up purposes. The microporous structure, alkaline pH and the inherent metal species of the alum sludge promoted the formation of metal sulphate species. This study demonstrated that alum sludge could be used as cost-effective, largely available, and efficient sorbent for H2S removal.

In-situ characterization of dissolved organic matter removal by coagulation using differential UV-Visible absorbance spectroscopy.

Removing dissolved organic matter (DOM) is of great concern due to its adverse effects on water supplies. Great effort has been given to studying DOM removal by coagulation, while the mechanism of DOM removal and the changes in its properties during coagulation have not been clearly illustrated due to the limitations of detection methods under practical environmental conditions. In this paper, the changes in DOM during coagulation were quantified using differential UV-Visible absorbance spectroscopy, and the differential spectra of DOM in the wavelength range of 200-600 nm could be deconvoluted into six Gaussian bands with maxima at approximately 200, 240, 276, 316, 385, and 457 nm after coagulation, respectively. The intensity of these maxima decreased with the type and dosage of coagulants. These observations should reflect the difference in the removability of DOM by coagulation, and this perspective was further confirmed by examining the deprotonation-protonation properties of DOM before and after coagulation. The affinity sites of DOM in coagulated waters, quantified by spectra parameter DlnA400 (differential log-transformed spectra at wavelength 400 nm) in combination with the revised NICA model, increased as the coagulant dosage, which indicates that coagulation is inclined to remove the DOM fraction with fewer functional groups. Polyaluminum chloride (PAC) and Al-aggregate (Al13) were more efficient than Alum for removing DOM due to their high efficiency for removing DOM fractions with fewer functional groups. The residual dissolved Al concentration depended on the total amount of reactive binding sites in DOM, and there was a strong linear correlation between residual dissolved Al and the total amount of reactive binding sites in DOM for Alum, while a weaker correlation was presented for PAC and Al13. This indicates that Ala was the dominant species to bind with the affinity sites in DOM to form residual dissolved Al.

Valorization of alum sludge via a pyrolysis platform using CO2 as reactive gas medium.

In an effort to seek a new technical platform for disposal of drinking water treatment sludge (DWTS: alum sludge), pyrolysis of DWTS was mainly investigated in this study. To establish a more sustainable thermolytic platform for DWTS, this study particularly employed CO2 as reactive gas medium. Thus, this study laid great emphasis on elucidating the mechanistic roles of CO2 during the thermolysis of DWTS. A series of the TGA tests of DWTS in CO2 in reference to N2 revealed no occurrence of the heterogeneous reaction between CO2 and the sample surface of DWTS. As such, at the temperature regime before initiating the Boudouard reaction (i.e., ≥700 °C), the mass decay patterns of DWTS in N2 and CO2 were nearly identical. However, the gaseous effluents from lab-scale pyrolysis of DWTS in CO2 in reference to N2 were different. In sum, the homogeneous reactions between CO2 and volatile matters (VMs) evolved from the thermolysis of DWTS led to the enhanced generation of CO. Also, CO2 suppressed dehydrogenation of VMs. Such the genuine mechanistic roles of CO2 in the thermolysis of DWTS subsequently led to the compositional modifications of the chemical species in pyrolytic oil. Furthermore, the biochar composite was obtained as byproduct of pyrolysis of DWTS. Considering that the high content of Al2O3 and Fe-species in the biochar composite imparts a strong affinity for As(V), the practical use of the biochar composite as a sorptive material for arsenic (V) was evaluated at the fundamental levels. This work reported that adsorption of As(V) onto the biochar composite followed the pseudo-second order model and the Freundlich isotherm model.

Recombinant hemagglutinin produced from Chinese Hamster Ovary (CHO) stable cell clones and a PELC/CpG combination adjuvant for H7N9 subunit vaccine development.

The novel H7N9 avian influenza A virus has caused human infections in China since 2013; some isolates from the fifth wave of infections have emerged as highly pathogenic avian influenza viruses. Recombinant hemagglutinin proteins of H7N9 viruses can be rapidly and efficiently produced with low-level biocontainment facilities. In this study, recombinant H7 antigen was obtained from engineered stable clones of Chinese Hamster Ovary (CHO) cells for subsequent large-scale production. The stable CHO cell clones were also adapted to grow in serum-free suspension cultures. To improve the immunogenicity of the recombinant H7 antigens, we evaluated the use of a novel combination adjuvant of PELC and CpG (PELC/CpG) to augment the anti-H7N9 immune responses in mice. We compared the effects with other adjuvants such as alum, AddaVax (MF59-like), and several Toll-like receptor ligands such as R848, CpG, and poly (I:C). With the PELC/CpG combination adjuvant, CHO cell-expressed rH7 antigens containing terminally sialylated complex type N-glycans were able to induce high titers of neutralizing antibodies in sera and conferred protection following live virus challenges. These data indicate that the CHO cell-expressed recombinant H7 antigens and a PELC/CpG combination adjuvant can be used for H7N9 subunit vaccine development.